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WO2002026991A2 - Gene 4 - Google Patents

Gene 4 Download PDF

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Publication number
WO2002026991A2
WO2002026991A2 PCT/EP2001/010998 EP0110998W WO0226991A2 WO 2002026991 A2 WO2002026991 A2 WO 2002026991A2 EP 0110998 W EP0110998 W EP 0110998W WO 0226991 A2 WO0226991 A2 WO 0226991A2
Authority
WO
WIPO (PCT)
Prior art keywords
gene
polynucleotide
dna
sequence
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2001/010998
Other languages
English (en)
Other versions
WO2002026991A3 (fr
Inventor
Colin Andrew Mclean Semple
Donald Robert Dunbar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Edinburgh
Medical Research Council
Akzo Nobel NV
Original Assignee
University of Edinburgh
Medical Research Council
Akzo Nobel NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Edinburgh, Medical Research Council, Akzo Nobel NV filed Critical University of Edinburgh
Priority to AU2002221623A priority Critical patent/AU2002221623A1/en
Priority to US10/399,157 priority patent/US20040053283A1/en
Priority to EP01985729A priority patent/EP1328642A2/fr
Publication of WO2002026991A2 publication Critical patent/WO2002026991A2/fr
Publication of WO2002026991A3 publication Critical patent/WO2002026991A3/fr
Anticipated expiration legal-status Critical
Priority to US11/847,173 priority patent/US20080102463A1/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals

Definitions

  • the present invention relates to a newly identified DNA sequence which is localized in the proximity of a breakpoint on chromosome 11 involved in a balanced t(l;l l)(q42.1;ql4.3) translocation.
  • the invention also relates to its encoded protein as well as to transformed cell lines.
  • Schizophrenia is a complex disease and the multifactorial and probable genetic heterogeneity of the condition complicates the application and interpretation of conventional linkage and association studies.
  • Psychiatric phenotypes linked with the translocation might be the result of position effects upon neighbouring genes on chromosome 11. It is therefor of importance to characterize genes on the chromosome 11 breakpoint region.
  • Glutamate is the major excitatory neurotransmitter in mammalian brain and is required for normal brain function, acting through a number of receptors. It has been hypothesised that hypoftinction of glutamatergic neurones has a pathological role in schizophrenia (Hirsch SR et al 1997, Pharmacol Biochem Behav 56 " :797-802), this hypothesis being strengthened by the fact that the NMDA receptor antagonist phencyclidine hydrochloride (TCP) can induce both positive and negative effects of schizophrenia. Reduced glutamate has been shown in schizophrenic cerebrospinal fluid and postmortem brain.
  • NAALADase activity has been shown to be decreased in schizophrenic brain (prefrontal and hippocampal regions), as have the products NAA and glutamate (Tsai G et al 1995, Arch Gen Psychiatry 52:829-836 ).
  • NAALADase for example 2-(phosphonomethyl)pentanedioic acid (2-PMPA or GPI5000) that have been proposed for treatment of conditions caused by excessive glutamate (i.e.
  • 2- PNPA was also tested in vivo in a rat stroke model (middle ear cerebral artery occlusion). The inhibitor was well tolerated at levels that produced a high degree of neuronal protection.
  • NAALADase agonists have not been discussed in the literature. By increasing the activity of NAALADase (using compounds that act on gene 4 protein) glutamate and NAA levels would be increased, and NAAG would be decreased (thus decreasing its inhibitory effect through mGluR3). In addition, NAALADase has also been shown to be a weak partial agonist of NMD A receptors (Pangalos MN et al 1999, J. Biol. Chem 274:8470-8483).
  • the present invention provides such a gene which is located on chromosome 11. More specific, the present invention provides for a gene, called gene 4 whose cDNA sequence is partially shown in SEQ ID NO:2.
  • sequences of the present invention can be used to prepare probes or as a source to prepare synthetic oligonucleotides to be used as primers in DNA amplification reactions allowing the isolation and identification of the complete gene.
  • the complete genetic sequence can be used in the preparation of vector molecules for expression of the protein in suitable host cells.
  • genes or variants thereof can be derived from cDNA or genomic DNA from natural sources or synthesized using known methods.
  • an additional embodiment of the invention is a method to isolate a gene comprising the steps of: a) hybridizing a DNA according to the present invention under stringent conditions against nucleic acids being RNA, (genomic) DNA or cDNA isolated preferably from tissues which highly express the DNA of interest; and b) isolating said nucleic acids by methods known to a skilled person in the art.
  • the tissues preferably are from human origin.
  • ribonucleic acids are isolated from brain.
  • the hybridization conditions are preferably highly stringent.
  • stringent means washing conditions of 1 x SSC, 0.1% SDS at a temperature of 65 °C; highly stringent conditions refer to a reduction in SSC towards 0.3 x SSC, preferably 0.1 x SSC.
  • the method to isolate the gene might comprise gene amplification methodology using primers derived from the nucleic acid according to the invention.
  • Complete cDNAs might also be obtained by combining clones obtained by e.g. hybridization with e.g. RACE cDNA clones.
  • the invention also includes the entire coding sequence part of which is indicated in SEQ ID NO: 2.
  • the invention also includes sequences coding for the same amino acid sequence as the amino acid sequence disclosed herein and presented in SEQ ID NO:l. Also portions of the coding sequence coding for individual domains of the expressed protein are part of the invention as well as allelic and species variations thereof.
  • a gene is expressed in a certain tissue as a splicing variant, resulting in an altered 5' or 3' mRNA or the inclusion of an additional exon sequence.
  • the messenger might have an exon less as compared to its counterpart.
  • the present invention provides for isolated polynucleotides encoding a glutamate carboxypeptidase, more specifically an N-acetyl-L-aspartyl-L- glutamate protease.
  • the DNA according to the invention may be obtained from cDNA.
  • the coding sequence might be genomic DNA, or prepared using DNA synthesis techniques.
  • the polynucleotide may also be in the form of RNA. If the polynucleotide is DNA, it may be in single stranded or double stranded form. The single strand might be the coding strand or the non-coding (anti-sense) strand.
  • the present invention further relates to polynucleotides which have at least 98% and even more preferably at least 99% identity with SEQ ID NO:2. Such polynucleotides encode polypeptides which retain the same biological function or activity as the natural, mature protein. Alternatively, also fragments of the above mentioned polynucleotides which code for domains of the protein which still are capable of binding to substrates are embodied in the invention.
  • the percentage of identity between two sequences can be determined with programs such as DNAMAN (Lynnon Biosoft, version 3.2). Using this program two sequences can be aligned using the optimal alignment algorithm of Smith and Waterman (1981, J. Mol. Biol, 747:195-197). After alignment of the two sequences the percentage identity can be calculated by dividing the number of identical nucleotides between the two sequences by the length of the aligned sequences minus the length of all gaps.
  • the DNA according to the invention will be very useful for in vivo or in vitro expression of the novel protease according to the invention in sufficient quantities and in substantially pure form.
  • the protein according to the invention comprises an amino acid sequence shown in SEQ ID NO: 1.
  • polypeptides according to the present invention include the polypeptides comprising variants of SEQ ID NO:l, i.e. polypeptides with a similarity of 98%, preferably 99% as compared to SEQ ID NO:l. Also portions of such polypeptides still capable of conferring biological effects are included. Especially portions which still bind to substrates form part of the invention. Such portions may be functional per se, e.g. in solubilized form or they might be linked to other polypeptides, either by known biotechnological ways or by chemical synthesis, to obtain chimeric proteins. Such proteins might be useful as therapeutic agent in that they may substitute the gene product in individuals with aberrant expression of the Gene 4 gene.
  • sequence of the gene may also be used in the preparation of vector molecules for the expression of the encoded protein in suitable host cells.
  • host cell and cloning vehicle combinations may be usefully employed in cloning the nucleic acid sequence coding for the Gene 4 protein of the invention or parts thereof.
  • useful cloning vehicles may include chromosomal, non-chromosomal and synthetic DNA sequences such as various known bacterial plasmids and wider host range plasmids and vectors derived from combinations of plasmids and phage or virus DNA.
  • Vehicles for use in expression of the genes or a part thereof comprising a peptidase activity containing domain of the present invention will further comprise control sequences operably linked to the nucleic acid sequence coding for the protein.
  • control sequences generally comprise a promoter sequence and sequences which regulate and/or enhance expression levels.
  • control and other sequences can vary depending on the host cell selected.
  • Suitable expression vectors are for example bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA. Vectors derived from chromosomal DNA are also included.
  • an origin of replication and/or a dominant selection marker can be present in the vector according to the invention.
  • the vectors according to the invention are suitable for transforming a host cell.
  • Recombinant expression vectors comprising the DNA of the invention as well as cells transformed with said DNA or said expression vector also form part of the present invention.
  • Suitable host cells according to the invention are bacterial host cells, yeast and other fungi, plant or animal host such as Chinese Hamster Ovary cells or monkey cells.
  • a host cell which comprises the DNA or expression vector according to the invention is also within the scope of the invention.
  • the engineered host cells can be cultured in conventional nutrient media which can be modified e.g. for appropriate selection, amplification or induction of transcription.
  • the culture conditions such as temperature, pH, nutrients etc. are well known to those ordinary skilled in the art.
  • the proteins according to the invention can be recovered and purified from recombinant cell cultures by common biochemical purification methods including ammonium sulfate precipitation, extraction, chromatography such as hydrophobic interaction chromatography, cation or anion exchange chromatography or affinity chromatography and high performance liquid chromatography. If necessary, also protein refolding steps can be included.
  • Gene 4 gene products according to the present invention can be used for the in vivo or in vitro identification of novel substrates or analogs thereof.
  • protease assay studies can be performed with cells transformed with DNA according to the invention or an expression vector comprising DNA according to the invention, said cells expressing the gene 4 gene products according to the invention.
  • the gene 4 gene product itself or the substrate-binding domains thereof can be used in an assay for the identification of functional substrates or analogs for the gene 4 gene products.
  • NAAG selectively activates the metabotropic glutamate receptor mGluR3 which in turn, decreases glutamate release.
  • a genomic BAG clone contiguous (contig) was made by searching previously mapped chromosome 11 genomic clone sequences against the publicly available High
  • PSMA accession number NM_004476
  • SEQ ID NO:2 A nucleotide acid sequence of part of the gene is shown in SEQ ID NO:2. The sequence codes for an amino acid sequence as identified in SEQ ID NO: 1.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne une séquence d'acides nucléiques codant une protéine présentant une activité de type NAALAdase. Selon les observations, cette activité est réduite dans les tissus du cerveau d'un sujet schizophrène. Sa protéine codée peut permettre de doser des modulateurs de glutamate peptidase.
PCT/EP2001/010998 2000-09-28 2001-09-21 Gene 4 Ceased WO2002026991A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2002221623A AU2002221623A1 (en) 2000-09-28 2001-09-21 Gene 4
US10/399,157 US20040053283A1 (en) 2000-09-28 2001-09-21 Gene 4
EP01985729A EP1328642A2 (fr) 2000-09-28 2001-09-21 Gene 4
US11/847,173 US20080102463A1 (en) 2000-09-28 2007-08-29 Gene 4

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP00308551.1 2000-09-28
EP00308551 2000-09-28

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/847,173 Continuation US20080102463A1 (en) 2000-09-28 2007-08-29 Gene 4

Publications (2)

Publication Number Publication Date
WO2002026991A2 true WO2002026991A2 (fr) 2002-04-04
WO2002026991A3 WO2002026991A3 (fr) 2002-11-28

Family

ID=8173291

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2001/010998 Ceased WO2002026991A2 (fr) 2000-09-28 2001-09-21 Gene 4

Country Status (4)

Country Link
US (2) US20040053283A1 (fr)
EP (1) EP1328642A2 (fr)
AU (1) AU2002221623A1 (fr)
WO (1) WO2002026991A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005040411A1 (fr) * 2003-10-17 2005-05-06 Bayer Healthcare Ag Diagnostics et therapeutiques pour maladies associees a la dipeptidase acide a liaison alpha n-acetylee de type 1 (naaladase de type 1)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6967315B2 (en) * 2002-06-12 2005-11-22 Steris Inc. Method for vaporizing a fluid using an electromagnetically responsive heating apparatus
US7547623B2 (en) * 2002-06-25 2009-06-16 Unitive International Limited Methods of forming lead free solder bumps

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0668777B2 (fr) * 1992-11-05 2011-02-09 Sloan-Kettering Institute For Cancer Research Antigene de membrane specifique a la prostate

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005040411A1 (fr) * 2003-10-17 2005-05-06 Bayer Healthcare Ag Diagnostics et therapeutiques pour maladies associees a la dipeptidase acide a liaison alpha n-acetylee de type 1 (naaladase de type 1)

Also Published As

Publication number Publication date
EP1328642A2 (fr) 2003-07-23
AU2002221623A1 (en) 2002-04-08
US20080102463A1 (en) 2008-05-01
US20040053283A1 (en) 2004-03-18
WO2002026991A3 (fr) 2002-11-28

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